1. Field of the Invention
This invention is related to the field of probe-based or primer-based target sequence detection, analysis and quantitation. More specifically, this invention relates to novel methods, kits and compositions pertaining to Detection Complexes wherein said methods, kits and compositions are used to generate detectable signal which is indicative of the presence, absence or quantity of one or more target sequences or target molecules of interest in a sample.
2. Description of the Related Art
Quenching of fluorescence signal can occur by either Fluorescence Resonance Energy Transfer xe2x80x9cFRETxe2x80x9d (also known as non-radiative energy transfer: See: Yaron et al., Analytical Biochemistry 95: 228-235 (1979) at p. 232, col. 1, lns. 32-39) or by non-FRET interactions (also known as radiationless energy transfer; See: Yaron et al., Analytical Biochemistry, 95 at p. 229, col. 2, lns. 7-13). The critical distinguishing factor between FRET and non-FRET quenching is that non-FRET quenching requires short range interaction by xe2x80x9ccollisionxe2x80x9d or xe2x80x9ccontactxe2x80x9d and therefore requires no spectral overlap between the moieties of the donor and acceptor pair (See: Yaron et al., Analytical Biochemistry 95 at p. 229, col. 1, lns. 22-42). Conversely, FRET quenching requires spectral overlap between the donor and acceptor moieties and the efficiency of quenching is directly proportional to the distance between the donor and acceptor moieties of the FRET pair (See: Yaron et al., Analytical Biochemistry, 95 at p. 232, col. 1, ln. 46 to col. 2, ln. 29). Extensive reviews of the FRET phenomenon are described in Clegg, R. M., Methods Enzymol., 221: 353-388 (1992) and Selvin, P. R., Methods Enzymol., 246: 300-334 (1995). Yaron et al. also suggested that the principles described therein might be applied to the hydrolysis of oligonucleotides (See: Yaron et al., Analytical Biochemistry, 95 at p. 234, col. 2, lns. 14-18).
The FRET phenomenon has been utilized for the direct detection of nucleic acid target sequences without the requirement that labeled nucleic acid hybridization probes or primers be separated from the hybridization complex prior to detection (See: Livak et al., U.S. Pat. No. 5,538,848). One method utilizing FRET to analyze Polymerase Chain Reaction (PCR) amplified nucleic acid in a closed tube format is commercially available from Perkin Elmer. The TaqMan(trademark) assay utilizes a nucleic acid hybridization probe which is labeled with a fluorescent reporter and a quencher moiety in a configuration which results in quenching of fluorescence in the intact probe. During the PCR amplification, the probe sequence specifically hybridizes to the amplified nucleic acid. When hybridized, the exonuclease activity of the Taq polymerase degrades the probe thereby eliminating the intramolecular quenching maintained by the intact probe. Because the probe is designed to hybridize specifically to the amplified nucleic acid, the increase in fluorescence intensity of the sample, caused by enzymatic degradation of the probe, can be correlated with the activity of the amplification process.
Nonetheless, this method preferably requires that each of the fluorophore and quencher moieties be located on the 3xe2x80x2 and 5xe2x80x2 termini of the probe so that the optimal signal to noise ratio is achieved (See: Nazarenko et al., Nucl. Acids Res., 25: 2516-2521 (1997) at p. 2516, col. 2, lns. 27-35). However, this orientation necessarily results in less than optimal fluorescence quenching because the fluorophore and quencher moieties are separated in space and the transfer of energy is most efficient when they are close. Consequently, the background emission from unhybridized probe can be quite high in the TaqMan(trademark) assay (See: Nazarenko et al., Nucl. Acids Res., 25: at p. 2516, col. 2, lns. 36-40).
The nucleic acid Molecular Beacon is another construct which utilizes the FRET phenomenon to detect target nucleic acid sequences (See: Tyagi et al., Nature Biotechnology, 14: 303-308 (1996)). A nucleic acid Molecular Beacon comprises a probing sequence embedded within two complementary arm sequences (See: Tyagi et al, Nature Biotechnology, 14: at p. 303, col. 1, lns. 22-30). To each termini of the probing sequence is attached one of either a fluorophore or quencher moiety. In the absence of the nucleic acid target, the arm sequences anneal to each other to thereby form a loop and hairpin stem structure which brings the fluorophore and quencher together (See: Tyagi et al., Nature Biotechnology, 14: at p. 304, col. 2, lns. 14-25). When contacted with target nucleic acid, the complementary probing sequence and target sequence will hybridize. Because the hairpin stem cannot coexist with the rigid double helix that is formed upon hybridization, the resulting conformational change forces the arm sequences apart and causes the fluorophore and quencher to be separated (See: Tyagi et al., Nature Biotechnology, 14: at p. 303, col. 2, lns. 1-17). When the fluorophore and quencher are separated, energy of the donor fluorophore does not transfer to the acceptor moiety and the fluorescent signal is then detectable. Since unhybridized xe2x80x9cMolecular Beaconsxe2x80x9d are non-fluorescent, it is not necessary that any excess probe be removed from an assay. Consequently, Tyagi et al. state that Molecular Beacons can be used for the detection of target nucleic acids in a homogeneous assay and in living cells. (See: Tyagi et al., Nature Biotechnology, 14: at p. 303, col. 2; lns. 15-77).
The arm sequences of the disclosed nucleic acid Molecular Beacon constructs are unrelated to the probing sequence (See: Tyagi et al., Nature Biotechnology, 14: at p. 303, col. 1; ln. 30). Because the Tyagi et al. Molecular Beacons comprise nucleic acid molecules, proper stem formation and stability is dependent upon the length of the stem, the G:C content of the arm sequences, the concentration of salt in which it is dissolved and the presence or absence of magnesium in which the probe is dissolved (See: Tyagi et al., Nature Biotechnology, 14: at p. 305, col. 1; lns. 1-16). Furthermore, the Tyagi et al. nucleic acid Molecular Beacons are susceptible to degradation by endonucleases and exonucleases.
Upon probe degradation, background fluorescent signal will increase since the donor and acceptor moieties are no longer held in close proximity. Therefore, assays utilizing enzymes known to have nuclease activity, will exhibit a continuous increase in background fluorescence as the nucleic acid Molecular Beacon is degraded (See: FIG. 7 in Tyagi et al: the data associated with (∘) and (xe2x96xa1) demonstrates that the fluorescent background, presumably caused by probe degradation, increases with each amplification cycle.) Additionally, nucleic acid Molecular Beacons will also, at least partially, be degraded in living cells because cells contain active nuclease activity.
The constructs described by Tyagi et al. are more broadly described in WO95/13399 (hereinafter referred to as xe2x80x9cTyagi2 et al.xe2x80x9d) except that Tyagi2 et al. also discloses that the nucleic acid Molecular Beacon may also be bimolecular wherein they define bimolecular as being unitary probes of the invention comprising two molecules (e.g. oligonucleotides) wherein half, or roughly half, of the target complement sequence, one member of the affinity pair and one member of the label pair are present in each molecule (See: Tyagi2 et al., p. 8, ln. 25 to p. 9, ln. 3). However, Tyagi2 et al. specifically states that in designating a unitary probe for use in a PCR reaction, one would naturally choose a target complement sequence that is not complementary to one of the PCR primers (See: Tyagi2 et al., p. 41, ln. 27). Assays of the invention include real-time and end-point detection of specific single-stranded or double stranded products of nucleic acid synthesis reactions, provided however that if unitary probes will be subjected to melting or other denaturation, the probes must be unimolecular (See: Tyagi2 et al., p. 37, lns. 1-9). Furthermore, Tyagi2 et al. stipulate that although the unitary probes of the invention may be used with amplification or other nucleic acid synthesis reactions, bimolecular probes (as defined in Tyagi2 et al.) are not suitable for use in any reaction (e.g. PCR) wherein the affinity pair would be separated in a target-independent manner (See: Tyagi2 et al., p. 13, lns. 9-12). Neither Tyagi et al. nor Tyagi2 et al. disclose, suggest or teach anything about PNA.
In a more recent disclosure, modified hairpin constructs which are similar to the Tyagi et al. nucleic acid Molecular Beacons, but which are suitable as primers for polymerase extension, have been disclosed (See: Nazarenko et al., Nucleic Acids Res., 25: 2516-2521(1997)). A method suitable for the direct detection of PCR-amplified DNA in a closed system is also disclosed. According to the method, the Nazarenko et al. primer constructs are, by operation of the PCR process, incorporated into the amplification product. Incorporation into a PCR amplified product results in a change in configuration which separates the donor and acceptor moieties. Consequently, increases in the intensity of the fluorescent signal in the assay can be directly correlated with the amount of primer incorporated into the PCR amplified product. The authors conclude, this method is particularly well suited to the analysis of PCR amplified nucleic acid in a closed tube format.
Because they are nucleic acids, the Nazarenko et al. primer constructs are admittedly subject to nuclease digestion thereby causing an increase in background signal during the PCR process (See: Nazarenko et al., Nucleic Acids Res., 25: at p. 2519, col. 1; lns. 28-39). An additional disadvantage of this method is that the Molecular Beacon like primer constructs must be linearized during amplification (See: Nazarenko et al., Nucleic Acids Res., 25: at p. 2519, col. 1, lns. 7-8). Consequently, the polymerase must read through and dissociate the stem of the hairpin modified Molecular Beacon like primer construct if fluorescent signal is to be generated. Therefore, the stem must be designed so that its stability does not inhibit the polymerase activity. Nazarenko et al. does not suggest, teach or disclose anything about PNA.
In still another application of FRET to target nucleic acid sequence detection, doubly labeled fluorescent oligonucleotide probes which have been rendered impervious to exonuclease digestion have also been used to detect target nucleic acid sequences in PCR reactions and in-situ PCR (See: Mayrand, U.S. Pat. No. 5,691,146). The oligonucleotide probes of Mayrand comprise a fluorescer (reporter) molecule attached to a first end of the oligonucleotide and a quencher molecule attached to the opposite end of the oligonucleotide (See: Mayrand, Abstract). Mayrand suggests that the prior art teaches that the distance between the fluorophore and quencher is an important feature which must be minimized and consequently the preferred spacing between the reporter and quencher moieties of a DNA probe should be 6-16 nucleotides (See: col. 7, lns. 8-24). Mayrand, however teaches that the reporter molecule and quencher moieties are preferably located at a distance of 18 nucleotides (See: col. 3, lns 35-36) or 20 bases (See: col. 7, lns. 25-46) to achieve the optimal signal to noise ratio. Consequently, both Mayrand and the prior art cited therein teach that the detectable properties of nucleic acid probes (DNA or RNA) comprising a fluorophore and quencher exhibit a strong dependence on probe length.
Resistance to nuclease digestion is also an important aspect of the invention (See: U.S. Pat. No. 5,691,146 at col. 6, lns. 42-64) and therefore, Mayrand suggests that the 5xe2x80x2 end of the oligonucleotide may be rendered impervious to nuclease digestion by including one or more modified internucleotide linkages (See: U.S. Pat. No. 5,691,146 at col. 6, lns. 45-50). Furthermore, Mayrand suggests that a polyamide nucleic acid (PNA) or peptide can be used as a nuclease resistant linkage to thereby modify the 5xe2x80x2 end of the oligonucleotide probe of the invention and render it impervious to nuclease digestion (See: U.S. Pat. No. 5,691,146 at col. 6, lns. 53-64). Mayrand does not however, disclose, suggest or teach that a PNA probe construct might be a suitable substitute for the practice of the invention despite having obvious knowledge of its existence. Furthermore, Mayrand does not teach one of skill in the art how to prepare and/or label a PNA with the fluorescer or quencher moieties.
The efficiency of energy transfer between donor and acceptor moieties as they can be influenced by oligonucleotide length (distance) has been further examined and particularly applied to fluorescent nucleic acid sequencing applications (See: Mathies et al., U.S. Pat. No. 5,707,804). Mathies et al. states that two fluorophores will be joined by a backbone or chain where the distance between the two fluorophores may be varied (See: U.S. Pat. No. 5,707,804 at col. 4, lns. 1-3). Thus, the distance must be chosen to provide energy transfer from the donor to the acceptor through the well-known Foerster mechanism (See: U.S. Pat. No. 5,707,804 at col. 4, lns. 7-9). Preferably about 3-10 nucleosides separate the fluorophores of a single stranded nucleic acid (See: U.S. Pat. No. 5,707,804 at col. 7, lns. 21-25). Mathies et al. does not suggest, teach or disclose anything about PNA.
From the analysis of DNA duplexes is has been observed that: 1: the efficiency of FET (or FRET as defined herein) appears to depend somehow on the nucleobase sequence of the oligonucleotide; 2: donor fluorescence changes in a manner which suggests that dye-DNA interactions affect the efficiency of FET; and 3: the Forster equation does not quantitatively account for observed energy transfer and therefore the length between donor and acceptor moieties attached to oligonucleotides cannot be quantitated, though it can be used qualitatively (See: Promisel et al., Biochemistry, 29: 9261-9268 (1990). Promisel et al. suggest that non-Forster effects may account for some of their observed but otherwise unexplainable results (See: Promisel et al., Biochemistry, 29: at p. 9267, col. 1, ln. 43 to p. 9268, col. 1, ln. 13). The results of promisel et al. suggest that the FRET phenomena when utilized in nucleic acids is not entirely predictable or well understood. Promisel et al. does not suggest, teach or disclose anything about PNA and, in fact, the manuscript predates the invention of PNA.
The background art thus far discussed does not disclose, suggest or teach anything about PNA oligomers to which are directly attached a pair of donor and acceptor moieties. In fact, the FRET phenomenon as applied to the detection of nucleic acids, appears to be confined to the preparation of constructs in which the portion of the probe which is complementary to the target nucleic acid sequence is itself comprised solely of nucleic acid.
FRET has also been utilized within the field of peptides. (See: Yaron et al. Analytical Biochemistry 95 at p. 232, col. 2, ln. 30 to p. 234, col. 1, ln. 30). Indeed, the use of suitably peptides as enzyme substrates appears to be the primary utility for peptides which are labeled with donor and acceptor pairs (See: Zimmerman et al., Analytical Biochemistry, 70: 258-262 (1976), Carmel et al., Eur. J. Biochem., 73: 617-625 (1977), Ng et al., Analytical Biochemistry, 183: 50-56 (1989), Wang et al., Tett. Lett., 31: 6493-6496 (1990) and Meldal et al., Analytical Biochemistry, 195: 141-147 (1991). Early work suggested that quenching efficiency of the donor and acceptor pair was dependent on peptide length (See: Yaron et al., Analytical Biochemistry 95 at p. 233, col. 2, lns. 36-40). However, the later work has suggested that efficient quenching was not so dependent on peptide length (See: Ng et al., Analytical Biochemistry, 183: at p. 54, col. 2, ln 23 to p. 55, col. 1, ln. 12; Wang et al., Tett. Lett., 31 wherein the peptide is eight amino acids in length; and Meldal et al. Analytical Biochemistry, 195 at p. 144, col. 1, lns. 33-37). It was suggested by Ng et al. that the observed quenching in long peptides might occur by an as yet undetermined mechanism (See: Ng et al., Analytical Biochemistry 183 at p. 55, cot. 1, ln 13 to col. 2, ln 7.)
Despite its name, peptide nucleic acid (PNA) is neither a peptide, a nucleic acid nor is it even an acid. Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide (pseudopeptide) which can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, 5,786,461 and Egholm et al., Nature 365: 566-568 (1993)). PNAs are synthesized by adaptation of standard peptide synthesis procedures in a format which is now commercially available. (For a general review of the preparation of PNA monomers and oligomers please see: Dueholm et al., New J. Chem., 21: 19-31 (1997) or Hyrup et. al., Bioorganic and Med. Chem. 4: 5-23 (1996)). Alternatively, labeled and unlabeled PNA oligomers can be purchased (See: PerSeptive Biosystems Promotional Literature: BioConcepts, Publication No. NL612, Practical PNA, Review and Practical PNA, Vol. 1, Iss. 2).
Being non-naturally occurring molecules, unmodified PNAs are not known to be substrates for the enzymes which are known to degrade peptides or nucleic acids. Therefore, unmodified PNAs should be stable in biological samples, as well as have a long shelf-life. Likewise, when complexed to a nucleic acid, PNAs shield the nucleic acid from degradation (See: WIPO patent application: Stanley et al., WO95/15974). Unlike nucleic acid hybridization which is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions which strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (Tomac et al., J. Am. Chem. Soc., 118: 5544-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (Egholm et al., Nature, at p. 566). However, the advantages in point mutation discrimination with PNA probes, as compared with DNA probes, in a hybridization assay appears to be somewhat sequence dependent (Nielsen et al., Anti-Cancer Drug Design 8: 53-65, (1993) and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)). As an additional advantage, PNAs hybridize to nucleic acid in both a parallel and antiparallel orientation, though the antiparallel orientation is preferred (See: Egholm et al., Nature at p. 566).
Despite the ability to hybridize to nucleic acid in a sequence specific manner, there are many differences between PNA probes and standard nucleic acid probes. These differences can be conveniently broken down into biological, structural, and physico-chemical differences. As discussed in more detail below, these biological, structural, and physico-chemical differences may lead to unpredictable results when attempting to use PNA probes in applications where nucleic acids have typically been employed. This non-equivalency of differing compositions is often observed in the chemical arts.
With regard to biological differences, nucleic acids, are biological materials that play a central role in the life of living species as agents of genetic transmission and expression. Their in vivo properties are fairly well understood. PNA, on the other hand is recently developed totally artificial molecule, conceived in the minds of chemists and made using synthetic organic chemistry. PNA has no known biological function and native (unmodified) PNA is not known to be a substrate for any polymerase, ligase, nuclease or protease.
Structurally, PNA also differs dramatically from nucleic acid. Although both can employ common nucleobases (A, C, G, T, and U), the backbones of these molecules are structurally diverse. The backbones of RNA and DNA are composed of repeating phosphodiester ribose and 2-deoxyribose units. In contrast, the backbones of the most common PNAs are composed of N-[2-(aminoethyl)]glycine subunits. Additionally, in PNA the nucleobases are connected to the backbone by an additional methylene carbonyl moiety.
PNA is not an acid and therefore contains no charged acidic groups such as those present in DNA and RNA. Because they lack formal charge, PNAs are generally more hydrophobic than their equivalent nucleic acid molecules. The hydrophobic character of PNA allows for the possibility of non-specific (hydrophobic/hydrophobic interactions) interactions not observed with nucleic acids. Further, PNA is achiral, providing it with the capability of adopting structural conformations the equivalent of which do not exist in the RNA/DNA realm.
The unique structural features of PNA result in a polymer which is highly organized in solution, particularly for purine rich polymers (See: Dueholm et al., New J. Chem., 21: 19-31 (1997) at p. 27, col. 2, lns. 6-30). Conversely, a single stranded nucleic acid is a random coil which exhibits very little secondary structure. Because PNA is highly organized, PNA should be more resistant to adopting alternative secondary structures (e.g. a hairpin stem and/or loop).
The physico/chemical differences between PNA and DNA or RNA are also substantial. PNA binds to its complementary nucleic acid more rapidly than nucleic acid probes bind to the same target sequence. This behavior is believed to be, at least partially, due to the fact that PNA lacks charge on its backbone. Additionally, recent publications demonstrate that the incorporation of positively charged groups into PNAs will improve the kinetics of hybridization (See: Iyer et al., J. Biol. Chem. 270: 14712-14717 (1995)). Because it lacks charge on the backbone, the stability of the PNA/nucleic acid complex is higher than that of an analogous DNA/DNA or RNA/DNA complex. In certain situations, PNA will form highly stable triple helical complexes through a process called xe2x80x9cstrand displacementxe2x80x9d. No equivalent strand displacement processes or structures are known in the DNA/RNA world.
Recently, the xe2x80x9cHybridization based screening on peptide nucleic acid (PNA) oligomer arraysxe2x80x9d has been described wherein arrays of some 1000 PNA oligomers of individual sequence were synthesized on polymer membranes (See: Weiler et al., Nucl. Acids Res., 25: 2792-2799(1997)). Arrays are generally used, in a single assay, to generate affinity binding (hybridization) information about a specific sequence or sample to numerous probes of defined composition. Thus, PNA arrays may be useful in diagnostic applications or for screening libraries of compounds for leads which might exhibit therapeutic utility. However, Weiler et al. note that the affinity and specificity of DNA hybridization to immobilized PNA oligomers depended on hybridization conditions more than was expected. Moreover, there was a tendency toward non-specific binding at lower ionic strength. Furthermore, certain very strong binding mismatches were identified which could not be eliminated by more stringent washing conditions. These unexpected results are illustrative of the lack of complete understanding of these newly discovered molecules (i.e. PNA).
In summary, because PNAs hybridize to nucleic acids with sequence specificity, PNAs are useful candidates for investigation as substitute probes when developing probe-based hybridization assays. However, PNA probes are not the equivalent of nucleic acid probes in both structure or function. Consequently, the unique biological, structural, and physico-chemical properties of PNA requires that experimentation be performed to thereby examine whether PNAs are suitable in applications where nucleic acid probes are commonly utilized.
This invention is directed to methods, kits and compositions which are used to detect the presence, absence or quantity of a target sequence and/or target molecule in a sample of interest. The preferred compositions of the invention are Detection Complexes, PCR Detection Complexes and Substrate Detection Complexes which are hybrids of at least two component polymers. At least two of the component polymers of the Detection Complex comprise at least one moiety from a set of donor and acceptor moieties, though the Detection Complex may comprise more than one set of donor and acceptor moieties and/or more than two component polymers. Component polymers are designed to form the Detection Complex by the interaction of interacting groups. Additionally, the Detection Complex may comprise one or more linkers and/or one or more spacer moieties as may be useful to construct a Detection Complex suitable for a particular application.
When the Detection Complex, PCR Detection Complex or Substrate Detection Complex is formed, at least one donor moiety of one component polymer is brought sufficiently close in space to at least one acceptor moiety of a second component polymer. Since the donor and acceptor moieties of the set of the assembled Detection Complex are closely situated in space, transfer of energy occurs between moieties of the set. When the Detection Complex dissociates, the donor and acceptor moieties do not interact sufficiently to cause substantial transfer of energy from the donor and acceptor moieties of the set. Consequently, Detection Complex formation/dissociation can be determined by measuring at least one physical property of at least one member of the set which is detectably different when the complex is formed as compared with when the component polymers of the Detection Complex, PCR Detection Complex or Substrate Detection Complex exist independently and unassociated.
The Detection Complexes and PCR Detection Complexes of this invention are primarily designed to dissociate as a direct or indirect consequence of the hybridization of one or more segments of a component polymer to a target sequence of a target molecule. Consequently, the Detection Complexes and PCR Detection Complexes can be used to detect the presence, absence or quantity of a target molecule of interest, which may be present in a sample of interest. The presence, absence or quantity of target molecule can then be determined by directly or indirectly correlating the dissociation of Detection Complex or PCR Detection Complex with the hybridization of a component polymer to the target sequence or priming site. Because the component polymers of a Detection Complex will preferably dissociate, the attached donor and acceptor moieties, which are independently attached to different polymers, can become far more separated in space as compared with unimolecular xe2x80x9cBeaconxe2x80x9d probes such as Molecular Beacons (PNA or nucleic acid) or Linear Beacons. As a consequence, the efficiency of energy transfer, which is proportional to the distance between the donor and acceptor moieties, will be far more substantially altered as compared with unimolecular probes wherein the donor and acceptor moieties are linked to the same polymer and therefore cannot be infinitely separated in space. Thus, the Detection Complexes and PCR Detection Complexes of this invention possess a substantial comparative advantage over unimolecular xe2x80x9cBeaconxe2x80x9d probes.
Though primarily designed to dissociate, the distance between donor and acceptor moieties may change merely because the probing segment of a probing polymer of a Detection Complex hybridizes to a target sequence whether or not the Detection Complex dissociates. Consequently, the energy transfer between donor and acceptor moieties of a set may be affected even though the Detection Complex does not dissociate provided there is a detectable change in at least one physical property of at least one member of a set which is detectably different in the native Detection Complex as compared with when the still intact Detection Complex is further complexed to a target sequence of a target molecule. Thus, the Detection Complexes of this invention may also be used to determine the presence absence or quantity of a target sequence or target molecule in a sample even though the Detection Complex does not dissociate.
Thus, in one embodiment, this invention is directed to Detection Complexes suitable for detecting or identifying the presence, absence or quantity of a target sequence and/or target molecule of interest in an assay. A Detection Complex comprises at least one probing polymer wherein the probing polymer has a probing segment which hybridizes to the target sequence, under suitable hybridization conditions, whether or not the Detection Complex dissociates. The probing polymer also has one or more interacting groups suitable for the formation of a complex with at least one other component polymer. The Detection Complex also comprises at least one annealing polymer which, at a minimum, has one or more interacting groups wherein the interaction of the interacting groups of the two or more component polymers form and stabilize the complex. The Detection Complex also comprises at least one set of donor and acceptor moieties. To each of at least two component polymers is linked at least one donor and one acceptor moiety such that formation of the complex facilitates transfer of energy between donor and acceptor moieties of each set in a manner which is detectably different from when the component polymers exist independently or unassociated or when the complex is free in solution as compared to when it is further complexed to a target sequence of a target molecule of interest. At least one of the component polymers of the Detection Complex is a non-nucleic acid polymer. The Detection Complex may exist in solution, may be immobilized to a support or may be one of two or more Detection Complexes arranged in an array.
ln still another embodiment, this invention is directed to non-nucleic acid polymers which are labeled with only a quencher but not a donor moiety. Preferably, the quencher is dabcyl. In preferred embodiments the non-nucleic acid polymer is terminally labeled with the quencher and most preferably the non-nucleic acid polymer is C-terminally labeled with dabcyl. Non-limiting, examples of several C-terminally dabcyl labeled PNAs are found in Table 1. For the examples listed in Table 1, the dabcyl moiety is conveniently linked to the N-E-amino group of the C-terminal lysine amino acid though this is not a limitation since other methods of terminal attachment exist.
ln still another embodiment, this invention is directed to Substrate Detection Complexes. Substrate Detection Complexes operate as a substrate for an enzyme to thereby generate changes in detectable signal in a target independent manner. A Substrate Detection Complex is very similar to the Detection Complexes hereinbefore described except the Substrate Detection Complex differs from a Detection Complex or PCR Detection Complex in that it does not contain a probing segment which hybridizes to a target sequence or priming site of a target molecule of interest. Thus, the Substrate Detection Complex does not directly interact with the target sequence or target molecule of interest. However, a Substrate Detection Complex, at a minimum, comprises at least two annealing polymers wherein at least one of the annealing polymers can interact with itself, another annealing polymer or another molecule in the assay to thereby form a substrate for an enzyme. The two or more annealing polymers further comprise interacting groups which form and stabilize the Substrate Detection Complex as well as linked donor and acceptor moieties.
The Detection Complexes, PCR Detection Complexes and Substrate Detection Complexes of this invention are suitable for detecting or identifying the presence, absence or quantity of a target sequence of a target molecule. Consequently, this invention is also directed to methods for the detection, identification or quantitation of a target sequence and/or target molecule in a sample.
In one embodiment, the method comprises contacting the sample with a Detection Complex or PCR Detection Complex and then detecting or identifying changes in detectable signal attributable to the transfer of energy between the donor and acceptor moieties of a Beacon Set upon hybridization or the probing segment of the probing polymer to the target sequence or upon direct or indirect dissociation of the complex. The signal detected can then be correlated with the presence, absence or quantity of the target sequence and/or target molecule in the sample. Generally, quantitation will involve comparison of the signal to a standard curve generated using a standardized assay and known quantities of target sequence and/or target molecule in representative samples.
In another embodiment, the method comprises forming the Detection Complex after the probing polymer or probing polymers have been allowed to interact with the target sequence or target molecule of interest. In this embodiment, the extent of formation of the Detection Complex can be measured by the change in detectable signal of at least one member of the Beacon Set before and after the formation of the Detection Complex. Since the amount of probing polymer or polymers and annealing polymer or polymers added to the sample can be controlled and calculated, the extent of formation of the Detection Complex, and the measurable change in detectable signal derived therefrom, can be used to determine the presence absence or quantity of a target sequence or target molecule in a sample of interest.
In still another embodiment, the Detection Complex is a substrate for an enzyme wherein the target molecule of interest is detected because the activity of the enzyme on the Substrate Detection Complex generates detectable signal in the presence of, or in proportion to, the presence or quantity of target molecule in the sample. The method comprises contacting the sample with probes and enzyme configured to generate target dependent enzyme activity. Generally, the assay is designed as a probe-based assay wherein at least one of the probes which complex with the target molecule is a probe-enzyme conjugate. The sample is then contacted with a Substrate Detection Complex and the changes in detectable signal attributable to the transfer of energy between the donor and acceptor moieties of a Beacon Set resulting from enzyme catalyzed dissociation of the complex are then measured. Generally, quantitation will involve comparison of the signal to a standard curve generated using a standardized assay and known quantities of target sequence and/or target molecule in representative samples.
In yet another embodiment, this invention is directed to a method for the formation of a Detection Complex, PCR Detection Complex or Substrate Detection Complex. Detection Complexes, PCR Detection Complexes and Substrate Detection Complexes are formed by mixing the two or more component polymers under conditions suitable for their interaction and assembly.
In still another embodiment, this invention is directed to kits which facilitate the useful practice of this invention. Thus, the preferred kits of this invention comprise one or more component polymers of a Detection Complex, PCR Detection Complex or Substrate Detection Complex and optionally other reagents useful for the practice of a method of this invention. Consequently, kits of this invention are suitable for detecting or identifying the presence, absence or quantity of a target sequence or target molecule which may be present in a sample of interest. As received by the end-user, the Detection Complex, PCR Detection Complex or Substrate Detection Complex may be preassembled or alternatively, the end-user may mix two or more of the component polymers to thereby generate the complex to be used with the kit.
In yet another embodiment, this invention is directed to a method for regenerating a support bound Detection Complex or an array of two or more support bound Detection Complexes. The method of regeneration comprises removing any hybridized target molecules from the surface and then contacting the surface with a quantity of at least one common labeled component polymer as is necessary to regenerate the one or many different Detection Complexes of the support or the array.
FIGS. 1A-1C illustrate several different embodiments of Detection Complexes.
FIG. 2 illustrates several different embodiments for the hybridization of a probing sequence of a Detection Complex with a target sequence.
FIGS. 3A-3C illustrate several different embodiments of Detection Complexes comprising multiple sets of detectable moieties.
FIGS. 4A and 4B illustrate the initial operation of PCR using a Detection Complex as a PCR primer.
FIG. 5 illustrates the operation of PCR in the first round wherein both the forward and reverse primers are Detection Complexes.
FIG. 6 illustrates the operation of PCR in the second round wherein both the forward and reverse primers are Detection Complexes.
FIG. 7 illustrates an amplicon prepared in a PCR amplification wherein both the forward and reverse primers are Detection Complexes.
FIG. 8A is a graphical illustration of fluorescence vs. temperature thermal profile for a Detection Complex assembled from two PNAs.
FIG. 8B is a graphical illustration of hybridization assay data for a Detection Complex assembled from two PNAs.
FIG. 9 is a graphical illustration of fluorescence vs. temperature thermal profile for a Detection Complex assembled from two PNAs.
FIGS. 10A and 10B are graphical illustrations of fluorescence vs. temperature thermal profiles for Detection Complexes assembled from a PNA and a DNA oligomer wherein the Detection Complexes operated as the forward and reverse primers in a PCR reaction.
FIG. 11 is a graphical illustration of tabular data obtained for a PCR reaction.
FIGS. 12A-12C are electronic composite negative images of photographs taken of a polyacrylamide gel used to analyze PCR reaction products for fluorescence.
FIG. 13 is a graphical illustration of tabular data obtained for a PCR reaction.
FIGS. 14A-14C are electronic composite negative images of photographs taken of a polyacrylamide gel used to analyze PCR reaction products for fluorescence.
FIG. 15 is a graphic illustration of data generated for a multiplex PCR assay using independently detectable PCR Detection Complexes.
FIGS. 16A and 16B are electronic composite negative images of photographs taken of a polyacrylamide gel used to analyze PCR reaction products for fluorescence.
FIGS. 17A and 17B are electronic composite negative images of photographs taken of a polyacrylamide gel used to analyze PCR reaction products for fluorescence.
FIG. 18 is an electronic composite negative image of two photographs taken of unopened PCR reaction sample tubes lying on a transilluminator.
FIG. 19A is a graphic illustration of tabular data obtained for a multiplex PCR assay.
FIG. 19B is an electronic composite negative of the image of a photograph of an ethidium bromide stained gel.
FIGS. 20A and 20B illustrate two different embodiments of a Substrate Detection Complex.
FIG. 21 is an illustration of a probe-based hybridization assay wherein a Substrate Detection Complex is used as a signal amplification process to detect the presence of the target molecule.